The present invention relates to an apparatus and method for the large-scale separation of a liquid from a solid that is dispersed therein on a continuous basis. More particularly, the apparatus and method provide a high-speed, centrifugal clarifier that contains a central, coaxial membrane surface that allows separation of a solid material irrespective of size and density from a liquid in a sterile environment, without the need for costly sterilization procedures, by use of a pre-sterilized disposable separation chamber.
Centrifugal clarifiers are well known devices for separating solids from a liquid-solid mixture and are used in a wide variety of applications such as separating sludge from oil and for harvesting cells from nutrient media in a biopharmaceutical fermentor. Biopharmaceutical manufacturing and research facilities use fermentors to produce high-density cultures of cells producing a useful product. The first step in recovering and purifying the product is separation of the cellular mass from the suspending growth media. The progression of the cultures are monitored for byproducts of cell growth in the media such as sucrose and carbon dioxide concentrations. Once the amounts of these substances reach a certain level, it indicates that the cells have stopped growing and should be harvested immediately or the cells may begin to die and release intracellular contaminants (endotoxins) upon further exposure to such conditions that begin to contaminate the product and further accelerate the rate of decline.
A high degree of sterility must be maintained in both the fermentor and the centrifugal clarifier. Introduction of contaminants could lead to damage or loss of the product and could prevent reuse of the potentially valuable cells.
The most commonly used clarification apparati are cross-flow membrane filtration devices, and large-volume, continuous flow centrifuges. For xe2x80x9cextra-cellularxe2x80x9d products (proteins that are secreted by the viable cell) filtration is generally used. Current filtration methodologies suffer from continual plugging of the membrane pores. As more and more cell-free filtrate is removed, the concentration of the retentate increases. This must be recirculated continuously at high velocities to prolong the time before the membrane fouls, or plugs; the concentration can never get so high as to avoid losing as much as 10% of the product in the fluid entrained in this recirculating stream. In addition to product loss, this high-shear cross flow to clear the membrane can result in product loss due to contamination, degradation, equipment fouling, and high shear-force-induced cell lysis. These events can cause downstream fouling of expensive purification equipment and processes, ultimately resulting in product loss or contamination. Finally, this technology suffers from the high cost of assuring the sterility of the apparatus through validation of clean-in-place or steam-in-place purification procedures of the product-contact surfaces by quality assurance organizations.
For xe2x80x9cintra-cellularxe2x80x9d products, the cells must first be lysed, or split open, by some mechanical means such as sonication, or by some chemical or enzyme that causes the cell wall to rupture, thereby releasing the product. Centrifuges are then used to separate the cellular debris from the suspending media containing the product. Unfortunately, the sludge-like sediment cakes onto the centrifuge surfaces and must later be manually scraped loose and collected for disposal. Furthermore, since the centrifuges are not sealed, the noise and aerosols that are created require that they be positioned within special enclosures. The aerosols can be hazardous. In addition, the supernatant from the centrifuge is often run through a secondary filter to remove contaminates that were too light to sediment and that may decrease the column life in the subsequent downstream processing steps.
Centrifuge equipment, like filtration, requires significant capital expenditures because they must also be designed to withstand the clean-in-place and steam-in-place procedures necessary to sterilize that step of the process. Extensive validation is required assure sterility. This validation requires a specialized staff that is expensive to train and maintain.
The decision between centrifuge and filtration is further complicated by the need to accurately predict clarification performance as the manufacturing scale of the process is increased. This process is carried out in several successively increasing scales. The cell strain is first perfected in xe2x80x9clab-scalexe2x80x9d fermenters that range in size from 10 to 100 liters. Then production experiments are run on xe2x80x9cpilot-scalexe2x80x9d fermentors that range in size, based on the cell strain involved, but are usually limited to 1,000 liters. After the initial production is shown to have produced clinically successful product, the process is scaled up to the long-term, xe2x80x9cproduction-scalexe2x80x9d fermenters which generally range from 10,000 to 100,000 liters. As the fermenter size is scaled up, it is critical that each step in the downstream process can also be scaled up with predictable results.
What is needed is an apparatus and method for separating a liquid from a solid dispersed therein which is scaleable and easy to use. What is further needed is a method and apparatus that eliminates the need for expensive sterility and validation procedures for clarification equipment used for harvesting cells from a fermentor.
See also: Tenthoff U.S. Pat. No. 4,411,645; Inge et al. U.S. Pat. No. 5,024,648; and Inge et al. U.S. Pat. No. 5,720,705.
The present invention provides a method and apparatus for separating a liquid from a solid dispersed therein.
More particularly the present invention provides a centrifugal clarifier that can incorporate a coaxial polishing filter and a method for separating a liquid from a liquid-solid mixture by rotation of a separation chamber. Further, the separation is carried out in a pre-sterilized and disposable container thus eliminating the need for clean-in-place or steam-in-place equipment, processes and validation. Still further, the present invention allows for the continuous introduction of a mixture into the centrifugal clarifier through a feed port and expulsion of clarified liquid into an outside container through an effluent port.
In the first embodiment, the centrifugal clarifier contains a cylindrical separation chamber that is covered by a lid with a hole therethrough. The chamber contains a pre-sterilized and disposable container that provides a sealed, sterile environment inside the clarifier. The lid of the disposable container fits into and is secured by the annular lid of the separation chamber and contains ports therethrough which allow introduction to and discharge from the separation chamber under sterile conditions. The disposable lid further contains a rotating face seal that provides a junction between the rotating and non-rotating components of the lid without leakage of aerosols or liquids. The seal faces are configured such that a sterile fluid can circulate through the seal to cool and lubricate the faces for high speed operation while insuring the sterility of the processing fluids.
A liquid-solid mixture is introduced through a feed port in the disposable lid and is carried to the bottom of the separation chamber through a feed tube. The particulate matter present in the mixture is sedimented to the walls of the chamber starting with the radially most outward and bottom position through rotation of the chamber, e.g., by an electric motor. The clarified liquid is then expelled into an outside container through an effluent port in the lid of the disposable container.
In a second embodiment, separation efficiency is enhanced by adding a coaxial filter within the disposable chamber such that the clarified liquid leaving through the effluent port is first passed through a membrane core. The membrane core is configured so as to prevent the smallest expected particle in the liquid-solid mixture from passing through the membrane while still allowing passage of the liquid. The membrane core rotates with the rotating chamber to minimize the relative rotational movement between the fluid (which is rotating at aproximately the same speed as the rotating chamber) and the membrane core. This eliminate any significant shear forces at the membrane surface which might lyse certain delicate cells such as mammalian cells. The synergy of combining centrifugation and filtration in series allows the centrifugal field that surrounds the membrane to be used as a coarse, pre-filtration clarification step that minimizes the cellular load impinging upon the membrane by sedimenting the particles radially away from the surface of the membrane. In turn, the membrane polishes the supernatant to a purity level that the centrifuge alone is incapable of achieving. The membrane is positioned in a manner that causes the cell-free, centrifugal supernatant to flow through the membrane material before exiting the centrifugal chamber, thus filtering out the floating or xe2x80x9cfoamingxe2x80x9d contaminants.
In a third embodiment, the membrane core is fixed and does not rotate. This presents significant shear forces at the membrane surface as the fluid, which is rotating at high speeds, approaches the membrane surface. This shear can be valuable tool in keeping the membrane clear when separating the more robust cellular material, such as yeast cells, which tend to stick to the membrane surface. In addition, since the lower seal has been eliminated, higher feed pressures can be used.
In another embodiment of the invention, the filter used in either the second or third embodiment above is designed to remove bacterial and viral contaminates from the product and, as such, is preferably a 0.2 micron membrane. This includes those contaminates that have been environmentally introduced by a flaw in the pre-sterilization procedures, or, more importantly, the larger intracellular viral contaminates that have been secreted during the fermentation process.
The throughput rate of a liquid-solid mixture into the centrifugal clarifier is adjustable over time as a function of the fill radius of the separation chamber. As solid material begins to accumulate in the separation chamber, the radius of the chamber will decrease and the throughput rate of the feed must consequently decrease. An appropriate throughput rate for the centrifugal clarifier can be determined mathematically.
Solid material collected in the pre-sterilized and disposable container may be stored in said container for use at a later time. Alternatively, the solid material may be resuspended in fresh liquid and pumped out through the feed port for immediate reuse.
The centrifugal clarifier of the invention is exemplary used as a first step of purification for products produced in a fermentor. It is envisioned that two devices will be required: A lab-scale clarifier for fermentors from 10 to 1,000 liters and one for full production scale of fermentors greater than 1,000 liters. The mechanical effects of separation on the cells and the throughput rates are mathematically determined and reliably accurate as the process is scaled up from xe2x80x9clab-scalexe2x80x9d fermenters that range in size from 10 to 100 liters to the long-term xe2x80x9cproduction-scalexe2x80x9d fermenters which generally range from 10,000 to 100,000 liters. As the fermenter size is scaled up, it is critical that each step in the downstream process can also be scaled up with predictable results, including the clarifier. Multiple clarifiers may be used in parallel to harvest the contents of fermentors larger than 1,000 liters. The size is ultimately limited to the size of the container of packed cells that must be easily removed from the clarifier. A 1,000 liter fermenter was arbitrarily selected as somewhere near the largest device. It typically yields 100 to 200 liters of packed cells weighing between 100-200 kilograms. This compares roughly with the size and weight of a standard 55-gallon drum, which is commonly used for the transport of materials in the pharma manufacturing facility. This can be handled conveniently by a davit affixed to the clarifier and by standard drum movers between the clarifier and the destination point.
It is envisioned that processing larger fermentors can be accomplished by running multiple clarifiers in parallel. Each positioned such that a common davit can remove the container of packed cells.
The clarifier is generally configured so as to be compact and portable, but the overall dimensions may vary depending on the particular application. Preferably it is mounted on swivel casters for extreme mobility. The structural elements of the clarifier are preferably constructed of common high-strength metals such as, e.g., aluminum, stainless steel, or composite materials.
Other aspects of the invention are discussed infra.